The cellular immune response against viruses involves cytotoxic T cell recognition of viral peptide fragments bound to class I MHC molecules. Class I molecules are heterodimers with a single binding site for peptide, which has been revealed by the crystal structure of a human class I molecule to be located on its top surface in a cleft between two long alpha-helices. The helices lie on top of an 8-stranded beta-sheet to form a peptide binding 'platform', a distinct structural domain predicted to be stable in isolation from the rest of the molecule. In the known structure, the peptide binding site is occupied, presumably by a mixture of endogenous peptides. In order to understand the physical forces governing the interaction between a peptide and class I molecule, it will be necessary to study binding of synthetic peptides to purified class I molecules, and make equimolar peptide-MHC complexes for crystallization to solve the x-ray structure of a specific peptide-MHC complex. These goals have not yet been achieved, because efforts to bind exogenous peptide to purified class I heterodimers have been unsuccessful, perhaps because of the presence of endogenous peptide in the binding site. This grant proposes to make isolated peptide binding platforms from which endogenous peptide can be removed, thereby allowing binding of exogenous peptide. Using a bacterial expression system, only the alpha 1 and alpha 2 domains of the heavy chain will be expressed to produce platforms, which will be denatured to remove endogenous peptide. As has been demonstrated for other single domain proteins, denatured platforms should refold correctly as facilitated by their single disulfide bond and monomeric nature (by contrast to class I heterodimers, in which denaturation is irreversible). Exogenous peptide will be added to the resulting 'empty' platforms for measurements of affinity and kinetic constants. Platforms will also be refolded in the presence of a synthetic T cell epitope peptide to make an equimolar peptide-MHC complex for crystallization. The structural integrity of empty platforms and platform-peptide complexes will be assayed by monoclonal antibody binding, resistance to protease digestion, and helical content in a circular dichroism spectrum. Platforms will be also directed to the surface of eukaryotic cells by transfection of platform genes linked to a lipid attachment signal, so that recognition by T cells can be assayed, as a test of their biological function. Computer models of MHC molecules will be used to choose residues to be mutated by site-directed mutagenesis, and mutant platforms will be tested for peptide binding ability and T cell recognition. The increase in knowledge gained by the ability to bind T cell epitope peptides to class I molecules can serve as a groundwork for the eventual design of peptides to serve as high affinity ligands for blocking the peptide binding sites of MHC molecules involved in autoimmune diseases.